Heparan sulfates belong to the glycosaminoglycan family of polysaccharides. They are present in most multicellular animals and have a ubiquitous distribution, being expressed on the cell surface and in the extracellular matrices (ECM) of most tissues (1, 2). Heparan sulfates usually exist as proteoglycans and there has been considerable progress in sequencing and cloning the core polypeptides of the molecule. So far, for example, at least eight different heparan sulfate proteoglycan (HSPG) core polypeptides have been identified on the cell surface (3).
Initially HSPGs were considered to play largely a structural role on the cell surface and in the ECM. However, heparan sulfate chains exhibit remarkable structural diversity (2, 4) which suggests that they may provide important signalling information for many biological processes. Thus, although heparan sulfate chains are initially synthesised as a simple alternating repeat of glucuronosyl and N-acetylglucosaminyl residues joined by .beta.1-4 and .alpha.1-4 linkages there are many subsequent modifications. The polysaccharide is N-deacetylated and N-sulfated and subsequently undergoes C5 epimerisation of glucuronosyl units to iduronosyl units, and various O-sulfations of the uronosyl and glucosaminyl residues. The variability of these modifications allows for some thirty different disaccharide sequences which, when arranged in different orders along the heparan sulfate chain, can theoretically result in a huge number of different heparan sulfate structures. In this regard, the anticoagulant polysaccharide heparin, present only in mast cell granules, represents an extreme form of heparan sulfate where epimerisation and sulfation have been maximised. Most heparan sulfates contain short stretches of highly sulfated residues joined by relatively long stretches of non-sulfated units.
There is now clear evidence that heparan sulfates play a critical role in a wide range of biological processes (2-4). In particular, they can act as ligands for adhesion molecules involved in cell--cell interactions (5, 6), participate in cell-ECM interactions (5, 6) and act as essential cell surface receptors for growth factors such as basic fibroblast growth factor (bFGF) (7, 8) and vascular endothelial growth factor (VEGF) (9). HSPGs are also a key component of basement membranes, which represent a major barrier to cell migration (10). Basement membrane barriers can only be breached when cells deploy a range of degradative enzymes (11) including an endoglycosidase, termed heparanase, which cleaves heparan sulfate chains (12, 13).
It has been shown that many of the biological processes in which heparan sulfates participate involve the recognition of unique heparan sulfate structures, with the position of the sulfates in the polysaccharide chain being of critical importance (3). For example, it has been demonstrated that defined heparan sulfate sequences are recognised by acidic and basic FGF (14-16) and cleaved by heparanases. Based on these observations, it has been an objective of the present inventors to synthesise sulfated oligosaccharides which block heparan sulfate recognition by growth factors, and inhibit cleavage of heparan sulfates by heparanases. In the case of blocking of growth factors, it was considered that low molecular weight mimics of heparan sulfate should be particularly effective, as it is now believed that cell surface heparan sulfates mediate the cross linking of growth factors bound to their receptors (17). Furthermore, sulfated oligosaccharides should be effective heparanase inhibitors by acting as non-cleavable substrates of this enzyme.
Sulfated oligosaccharides with growth factor inhibitory activity have a number of clinical uses. Heparin/heparan sulfate binding growth factors, such as bFGF and VEGF, are potent inducers of angiogenesis (18). In adults, angiogenesis is a relatively rare occurrence except during wound healing. However, there are a number of "angiogenesis-dependent diseases" in adults where angiogenesis is critically important (18-20). The most important of these is the angiogenesis associated with the growth of solid tumours, proliferative retinopathies and rheumatoid arthritis. Sulfated oligosaccharides which blocked the action of key angiogenic growth factors, such as bFGF and VEGF, would be particularly useful for the treatment of these angiogenesis-dependent diseases.
Similarly, sulfated oligosaccharides which inhibit heparanase action have a number of clinical applications. The subendothelial basement membrane represents a major physical barrier for the passage of endothelial cells, tumour cells and leukocytes through the blood vessel wall. The heparanase enzyme, combined with a range of proteolytic enzymes (eg, plasmin, matrix metalloproteinases), plays an essential part in basement membrane degradation by invading cells (11-13, 21). Thus, by preventing basement membrane degradation, sulfated oligosaccharides with heparanase-inhibitory activity should exhibit anti-metastatic and anti-inflammatory activity, and in addition may inhibit early stages of angiogenesis. The use of sulfated oligosaccharides which simultaneously inhibit angiogenic growth factor action and the heparanase enzyme would be preferred in many clinical situations, eg, treatment of highly metastatic solid tumours and rheumatoid arthritis.
Prior International Patent Application No. PCT/AU88/00017 (Publication No. WO 88/05301) discloses the use of sulfated polysaccharides such as heparin and modified heparin, fucoidin, pentosan sulfate, dextran sulfate and carrageenin lambda, which block or inhibit heparanase activity, in anti-metastatic and/or anti-inflammatory treatment of an animal or human patient.
In work leading to the present invention, the inventors have prepared sulfated oligosaccharides using either naturally occurring oligosaccharides or totally synthetic oligosaccharides comprising hexose-containing homopolymers. Some of these compounds have been demonstrated to be potent inhibitors of human angiogenesis, tumor metastatis and inflammation. The data obtained is consistent with the sulfated oligosaccharides exhibiting their biological effects by inhibiting angiogenic growth factor action and/or heparanase function, and certain sulfated oligosaccharides have been obtained which are potent inhibitors of both angiogenesis and heparanase activity.